Urinary porphyrin excretion in children with mercury amalgam treatment: findings from the Casa Pia Children's Dental Amalgam Trial.

Increases in the urinary concentrations of pentacarboxyl- and coproporphyrins and the appearance of the atypical precoproporphyrin have been defined in relation to mercury (Hg) body burden in animal studies, and this change in the porphyrin excretion pattern has been described as a biomarker of occupational Hg exposure and toxicity in adult human subjects. In the present studies, urinary porphyrins were determined in relation to Hg exposure in children and adolescents, 8-18 yr of age, over the 7-yr course of a clinical trial designed to evaluate the neurobehavioral and renal effects of dental amalgam in children. Subjects were randomized to either dental amalgam or composite resin treatments. Urinary porphyrins and creatinine concentrations were measured at baseline and annually in all subjects. Results were evaluated using linear regression analysis. No significant differences between treatment groups (amalgam versus composite) were found when comparing all subjects for any of the porphyrins of interest. However, incipent amalgam treatment-specific increases were observed in the mean concentrations of penta-, precopro- and coproporphyrins especially when the analyses were restricted to younger subjects (8 to 9 yr old at baseline), and these increases were most apparent during yr 2 through 3 of follow-up, the period of highest mercury exposure from amalgam treatment. Based on the mean number of amalgam fillings received by children in this group (17.8), the renal Hg concentration associated with incipient increases in urinary porphyrins was estimated to be approximately 2.7 microg/g renal cortex. This value corresponds to an observed mean urinary Hg concentration of 3.2 microg/g creatinine, which is approximately fivefold less than that at which renal damage from Hg exposure is estimated to occur in children. These findings are consistent with growing evidence supporting the sensitivity of urinary porphyrins as a biological indicator of subclinical Hg exposure in children.

The contribution of dental amalgam to urinary mercury excretion in children.

BACKGROUND: Urinary mercury concentrations are widely used as a measure of mercury exposure from dental amalgam fillings. No studies have evaluated the relationship of these measures in a longitudinal context in children. OBJECTIVE: We evaluated urinary mercury in children 8-18 years of age in relation to number of amalgam surfaces and time since placement over a 7-year course of amalgam treatment. METHODS: Five hundred seven children, 8-10 years of age at baseline, participated in a clinical trial to evaluate the neurobehavioral effects of dental amalgam in children. Subjects were randomized to either dental amalgam or resin composite treatments. Urinary mercury and creatinine concentrations were measured at baseline and annually on all participants. RESULTS: Treatment groups were comparable in baseline urinary mercury concentration (approximately 1.5 microg/L). Mean urinary mercury concentrations in the amalgam group increased to a peak of approximately 3.2 microg/L at year 2 and then declined to baseline levels by year 7 of follow-up. There was a strong, positive association between urinary mercury and both number of amalgam surfaces and time since placement. Girls had significantly higher mean urinary mercury concentrations than boys throughout the course of amalgam treatment. There were no differences by race in urinary mercury concentration associated with amalgam exposure. CONCLUSIONS: Urinary mercury concentrations are highly correlated with both number of amalgam fillings and time since placement in children. Girls excrete significantly higher concentrations of mercury in the urine than boys with comparable treatment, suggesting possible sex-related differences in mercury handling and susceptibility to mercury toxicity.

Attenuation of nuclear factor kappa B (NF-kappaB) promotes apoptosis of kidney epithelial cells: a potential mechanism of mercury-induced nephrotoxicity.

Nuclear factor kappa B (NF-kappaB), a pleiotropic transcriptional factor that promotes cell survival and protects cells from apoptosis, requires reduced thiols at critical steps in its activation pathway. Mercuric ion (Hg(2+)), one of the strongest thiol-binding agents known, impairs NF-kappaB activation and transcriptional activity in normal rat kidney epithelial (NRK52E) cells at concentrations as low as 0.5 microM by binding to specific reduced thiol moieties in the NF-kappaB activation pathway. We hypothesized that prevention of NF-kappaB activation by Hg(2+) will increase the sensitivity of kidney cells to the apoptosis-inducing effects of other toxicants to which these cells are otherwise resistant by virtue of their NF-kappaB-activating capacity. Fewer than 5% of untreated kidney cells in culture (70-90% confluent) were found to be apoptotic when evaluated by DNA fragmentation (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling) or flow cytometric DNA profile analyses. Hg(2+) (5 microM) treatment for 24 hr increased this proportion by 1.5- to 2-fold. Neither lipopolysaccharide (LPS) (1 microg/mL) nor tumor necrosis factor-alpha (TNF-alpha; 300 U/mL), both potent activators of NF-kappaB in kidney cells, significantly altered the proportion of apoptotic cells, compared with untreated controls, when applied without Hg(2+) pretreatment. However, when LPS or TNF-alpha was administered after Hg(2+) pretreatment (5 microM for 30 min), the proportion of cells undergoing apoptosis 22 hr later increased by 4- to 6-fold compared with untreated controls. In contrast, Hg(2+) pretreatment did not increase the amount of apoptosis caused by apoptosis-inducing agents that do not activate NF-kappaB (staurosporine, Fas ligand). These findings suggest that Hg(2+) enhances the sensitivity of kidney cells to apoptotic stimuli as a consequence of inhibition of NF-kappaB activity. Because apoptosis is known to play a key role in the pathogenesis of renal failure resulting from toxicant injury to proximal tubular cells, promotion of apoptosis via inhibition of NF-kappaB activity may define a novel molecular mechanism by which Hg(2+) toxicity is initiated in kidney cells.

Nuclear factor kappaB activity determines the sensitivity of kidney epithelial cells to apoptosis: implications for mercury-induced renal failure.

Nuclear factor kappa B (NF-kappaB) is a thiol-dependent transcriptional factor that promotes cell survival and protects cells from apoptotic stimuli. Numerous studies have demonstrated increased sensitivity to apoptosis associated with inhibition of NF-kappaB activation in various cell types. We have previously demonstrated that mercuric ion (Hg(2+)), one of the strongest thiol-binding agents known, impairs NF-kappaB activation and DNA binding at low microM concentrations in kidney epithelial cells. In the present studies we investigated the hypothesis that inhibition of NF-kappaB activation by Hg(2+) and other selective NF-kappaB inhibitors would increase the sensitivity of kidney epithelial (NRK52E) cells to apoptogenic agents to which these cells are normally resistant. Fewer than 10% of untreated cells in culture were found to be apoptotic when evaluated by DNA fragmentation (TUNEL) assay. Treatment of cells with Hg(2+) in concentrations up to 5 microM or with tumor necrosis factor-alpha (TNF) (300 units/ml) did not significantly increase the proportion of apoptotic cells, compared with untreated controls. However, when TNF was given following Hg(2+) pretreatment (0.5 to 5 microM for 30 min), the proportion of cells undergoing apoptosis increased by 2- to 6-fold over that seen in untreated controls. Kidney cells pretreated with specific NF-kappaB inhibitors (Bay11-7082 or SN50) prior to TNF also showed a significant increase in apoptosis. Increased sensitivity to apoptotic cell death following these treatments was significantly attenuated in cells transfected with a p65 expression vector. In studies in vivo, rats pretreated by intraperitoneal injection with Hg(2+) (0.75 mg/kg) 18 h prior to administration of bacterial lipopolysaccharide (LPS) (10 mg/kg) displayed impaired NF-kappaB activation and an increased mitochondrial cytochrome c release in kidney cortical cells. These findings are consistent with the view that prevention of NF-kappaB activity in vitro or in vivo enhances the sensitivity of kidney cells to apoptotic stimuli to which these cells are otherwise resistant. Since apoptosis is known to play a seminal role in the pathogenesis of renal failure caused by toxicant injury to tubular cells, the present findings suggest that inhibition of NF-kappaB activity may define a molecular mechanism underlying the pathogenesis of Hg(2+) toxicity in kidney cells.

Urinary porphyrin excretion in neurotypical and autistic children.

BACKGROUND: Increased urinary concentrations of pentacarboxyl-, precopro- and copro-porphyrins have been associated with prolonged mercury (Hg) exposure in adults, and comparable increases have been attributed to Hg exposure in children with autism (AU). OBJECTIVES: This study was designed to measure and compare urinary porphyrin concentrations in neurotypical (NT) children and same-age children with autism, and to examine the association between porphyrin levels and past or current Hg exposure in children with autism. METHODS: This exploratory study enrolled 278 children 2-12 years of age. We evaluated three groups: AU, pervasive developmental disorder-not otherwise specified (PDD-NOS), and NT. Mothers/caregivers provided information at enrollment regarding medical, dental, and dietary exposures. Urine samples from all children were acquired for analyses of porphyrin, creatinine, and Hg. Differences between groups for mean porphyrin and Hg levels were evaluated. Logistic regression analysis was conducted to determine whether porphyrin levels were associated with increased risk of autism. RESULTS: Mean urinary porphyrin concentrations are naturally high in young children and decline by as much as 2.5-fold between 2 and 12 years of age. Elevated copro- (p < 0.009), hexacarboxyl- (p < 0.01) and pentacarboxyl- (p < 0.001) porphyrin concentrations were significantly associated with AU but not with PDD-NOS. No differences were found between NT and AU in urinary Hg levels or in past Hg exposure as determined by fish consumption, number of dental amalgam fillings, or vaccines received. CONCLUSIONS: These findings identify disordered porphyrin metabolism as a salient characteristic of autism. Hg exposures were comparable between diagnostic groups, and a porphyrin pattern consistent with that seen in Hg-exposed adults was not apparent.

Urinary porphyrin excretion in normal children and adolescents.

BACKGROUND: Urinary porphyrins are diagnostic of various metabolic disorders and xenobiotic exposures, but comprehensive normative data for urinary porphyrin concentrations in children are currently unavailable. METHODS: Subjects were participants in a prospective, randomized, controlled clinical trial of dental materials safety, 8 to 12 y at inception, who were followed longitudinally for 7 y after baseline with an extensive battery of neurobehavioral, neurological, renal function and urinary porphyrin assessments. Porphyrins were quantified by HPLC. Linear regression analyses were used to measure associations of porphyrin levels with age and gender. RESULTS: Mean concentrations, 95% confidence intervals, and 10th, 50th, and 90th percentiles for all 5 typically excreted urinary porphyrins are presented by year of age and by gender. Unadjusted urinary concentrations (microg/l) of all 5 porphyrins remained relatively constant throughout the age range of 8-18 y for both males and females. In contrast, creatinine-adjusted urinary porphyrin concentrations (microg/g) declined significantly throughout this age range in both genders. Boys had significantly higher pentacarboxyl- and copro-porphyrin levels compared with girls both before and after creatinine adjustment. CONCLUSIONS: Normative longitudinal data provided herein may facilitate the clinical assessment of pediatric metabolic disorders and may be of particular relevance in evaluating porphyrin changes as a biological indicator of disease or xenobiotic exposures among children and adolescents.

RhoA regulation of NF-kappaB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells.

Numerous studies have demonstrated a central role of renal tubular epithelial cells in the etiology of kidney injury and disease through the elaboration of inflammatory mediators. However, little is known about the cellular signaling mechanisms involved in this process. In this study we employed normal rat kidney epithelial (NRK52E) cells to identify a novel LPS-induced signaling pathway in which RhoA-mediated AP-1 activity promotes expression of cyclooxygenase-2 (COX-2) with consequent feedback inhibition of NF-kappaB activation through IKKbeta. Inhibition of RhoA signaling using either the RhoA kinase inhibitor Y-27632 or a dominant negative mutant of RhoA (RhoA-DN) dramatically extended the duration of p65-DNA binding, IkappaBalpha phosphorylation, and IKKbeta activity following LPS treatment. Prolongation of events associated with NF-kappaB activation was also observed in cells pretreated and/or cotransfected with the JNK inhibitor SP600125 or deletion mutants of MEKK1 (MEKK1-KD) or Jun (Jun-DN). Conversely, constitutive expression of RhoA prevented NF-kappaB activation by LPS, and this effect was reversed by cotransfection with MEKK1-KD. In addition, we found that the RhoA/AP-1 signaling axis plays a necessary role in COX-2 expression by LPS and that this effect is independent of NF-kappaB activation. Moreover, inhibition of COX-2 activity results in persistent p65-DNA binding, IkappaBalpha phosphorylation, and IKKbeta activity, similar to that observed after prevention of RhoA/AP-1 axis signaling. These findings suggest that COX-2 links the RhoA/AP-1 signaling cascade to NF-kappaB activation, thereby defining a novel integrated model for regulation of the inflammatory response of kidney epithelial cells to LPS and potentially other external stimuli.

The association between genetic polymorphisms of coproporphyrinogen oxidase and an atypical porphyrinogenic response to mercury exposure in humans.

Previous studies have demonstrated highly specific urinary porphyrin profile (UPP) changes in response to mercury (Hg) exposure in animals and human subjects and have defined the biochemical etiology of this effect as selective alteration of the heme pathway enzymes, uroporphyrinogen decarboxylase (UROD), and coproporphyrinogen oxidase (CPOX) by Hg in the kidney. Ongoing validation studies in a population of dental practitioners with low-level occupational Hg exposure have demonstrated the predicted UPP change among approximately 85% of subjects. This study focused on the genetic etiology of an atypical porphyrinogenic response (APR) seen among the remaining 15% of Hg-exposed subjects, characterized by excess excretion of 4- and 5-carboxyl porphyrins and also of the atypical ketoisocoproporphyrin (KICP). Automated DNA-sequencing-based assays were developed to examine the 7 exons and flanking intron-exon boundaries of the CPOX gene. Among several polymorphisms identified, an A814C variant in exon 4 encoding a N272H substitution was found to be predominant among subjects with the APR. Studies suggest that this variant CPOX preferentially converts the upstream 5-carboxylporphyrin (5-CP) to KICP. By partially inhibiting the 5- to 4-decarboxylation step of UROD, Hg promotes 5-CP accumulation, accounting for e xcess KICP excretion and the APR in Hg-exposed subjects carrying the variant CPOX gene. This finding represents the first report of a polymorphism in a human gene that modifies the effect of Hg on a biological process. The APR might serve as a biomarker of both Hg exposure and susceptibility to Hg toxicity.